Inducing Secondary Metabolite Production by the Endophytic Fungus

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Inducing Secondary Metabolite Production by the Endophytic Fungus Fusarium tricinctum through Coculture with Bacillus subtilis Antonius R. B. Ola,†,‡ Dhana Thomy,† Daowan Lai,*,† Heike Brötz-Oesterhelt,† and Peter Proksch*,† †

Institut für Pharmazeutische Biologie und Biotechnologie, Heinrich-Heine-Universität Düsseldorf, Universitätsstrasse 1, Geb. 26.23, 40225 Düsseldorf, Germany ‡ Department of Chemistry, Faculty of Science and Engineering, Nusa Cendana University, Jalan Adisucipto Penfui, 85001 Kupang, Indonesia S Supporting Information *

ABSTRACT: Coculturing the fungal endophyte Fusarium tricinctum with the bacterium Bacillus subtilis 168 trpC2 on solid rice medium resulted in an up to 78-fold increase in the accumulation in constitutively present secondary metabolites that included lateropyrone (5), cyclic depsipeptides of the enniatin type (6−8), and the lipopeptide fusaristatin A (9). In addition, four compounds (1−4) including (−)-citreoisocoumarin (2) as well as three new natural products (1, 3, and 4) were not present in discrete fungal and bacterial controls and only detected in the cocultures. The new compounds were identified as macrocarpon C (1), 2-(carboxymethylamino)benzoic acid (3), and (−)-citreoisocoumarinol (4) by analysis of the 1D and 2D NMR and HRMS data. Enniatins B1 (7) and A1 (8), whose production was particularly enhanced, inhibited the growth of the cocultivated B. subtilis strain with minimal inhibitory concentrations (MICs) of 16 and 8 μg/mL, respectively, and were also active against Staphylococcus aureus, Streptococcus pneumoniae, and Enterococcus faecalis with MIC values in the range 2−8 μg/mL. In addition, lateropyrone (5), which was constitutively present in F. tricinctum, displayed good antibacterial activity against B. subtilis, S. aureus, S. pneumoniae, and E. faecalis, with MIC values ranging from 2 to 8 μg/mL. All active compounds were equally effective against a multiresistant clinical isolate of S. aureus and a susceptible reference strain of the same species.

B

strains would seem to greatly expand the possibility to discover new bioactive molecules. Several coculture studies have been reported in the past that resulted either in an enhanced production of known metabolites or in the discovery of new molecules. Cocultivation of two marine microorganisms, the fungus Emericella sp. and the actinomycete Salinispora arenicola, enhanced the expression of the emericellamide biosynthesis gene cluster 100-fold.5 The production of new cytotoxic diterpenes, libertellenones A−D, was induced when the marine-derived fungus Libertella sp. was cocultivated with the marine bacterium CNJ-328.8 Another example was the production of a new antibiotic, pestalone, from a coculture of the marine fungus Pestalotia sp. with an unknown α-proteobacterium, CNJ-328.9

acteria and fungi co-inhabit a wide variety of habitats such as soil, water, or the living tissues of higher plants, where they may be present as endophytes.1,2 One important interaction of fungi and bacteria includes competition for limited nutrients, which is known as a major ecological factor that triggers natural product biosynthesis and accumulation in prokaryotes and eukaryotes alike.3,4 Coculturing of different microbes rather than maintaining axenic cultures as is usually practiced in microbiology forces direct interactions that may enhance the accumulation of constitutively present natural products5−7 or may trigger the expression of silent biosynthetic pathways yielding new compounds.8,9 Recent findings show that intimate physical interaction during coculture of the fungus Aspergillus nidulans and the bacterium Streptomyces rapamycinicus triggered the expression of gene clusters for orselinic acid and lecanoric acid that are kept silent under standard laboratory conditions.6,7 Therefore, an induction of silent biosynthetic pathways by coculturing of two or more different microbial © XXXX American Chemical Society and American Society of Pharmacognosy

Received: July 19, 2013

A

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Figure 1. HPLC chromatograms of the EtOAc extracts from coculture experiments detected at UV 235 nm: (a) control of B. subtilis, (b) control of F. tricinctum, (c) control of S. lividans, (d) coculture of F. tricinctum with S. lividans (*unidentified metabolites), (e) coculture of F. tricinctum with B. subtilis.

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In this paper, we report the influence of Bacillus subtilis 168 trpC2 on the secondary metabolism of Fusarium tricinctum during cocultivation. We found an up to 78-fold increase in the accumulation of constitutively present fungal products such as lateropyrone (5), cyclic depsipeptides of the enniatin type (6− 8), and the lipopeptide fusaristatin A (9) in the presence of B. subtilis compared to axenic cultures of F. tricinctum. In addition, four metabolites (1−4) including three new natural products, 1, 3, and 4, were identified only in cocultures (Figure 1). Interestingly, when F. tricinctum was cocultured with a second bacterium (Streptomyces lividans), compounds 1−4 were not detected, whereas the accumulation of other, yet unidentified compounds was induced that were lacking in cocultures of F. tricinctum and B. subtilis. This finding suggests that the response of F. tricinctum toward different bacteria may be a specific rather than a general reaction toward prokaryotes.

RESULTS AND DISCUSSION

The strain of F. tricinctum used in this study had been isolated previously as an endophytic fungus from the plant Aristolochia paucinervis.10 The bacterial species, B. subtilis, that was used for cocultivation with F. tricinctum is known from different habitats including soil but was also isolated from higher plants.11−13 Thus, F. tricinctum and B. subtilis can both be considered as endophytes and hence may even be suspected to co-occur in plants even though no direct evidence for this hypothesis has been brought forward so far. F. tricinctum is known to produce bioactive cyclic peptides of the enniatin type, which are among the characteristic constituents of this species.10,14 When F. tricinctum was cultured axenically on solid rice medium, average yields of the leading enniatin derivatives per culture flask were 8.78 mg for enniatin B (6), 2.44 mg for enniatin B1 (7), and 0.28 mg for enniatin A1 (8) (Table 1). Coculturing of F. Table 1. Yield of Induced Metabolites per Flask during Coculture of F. tricinctum and B. subtilis (n = 8) vs Axenic Controls of F. tricinctum (n = 8) compound 1 2 3 4 5 6 7 8 9 a

B

control (mg) a

n.d. n.d. n.d. n.d. 3.40 ± 0.94 8.78 ± 1.54 2.44 ± 1.32 0.28 ± 0.15 10.05 ± 1.24

coculture (mg)

increase (fold)

± ± ± ± ± ± ± ± ±

1.4 9.0 36.3 78.0 7.9

0.71 3.86 15.99 0.36 4.85 79.58 88.54 21.85 79.40

0.19 1.52 1.83 0.07 2.32 21.85 24.42 8.35 28.54

n.d.: not detected. dx.doi.org/10.1021/np400589h | J. Nat. Prod. XXXX, XXX, XXX−XXX

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tricinctum and B. subtilis resulted in a strong enhancement of enniatin accumulation. Average production of enniatins per flask in cocultures reached 79.58 mg for enniatin B (6), 88.54 mg for enniatin B1 (7), and 21.85 mg for enniatin A1 (8), which accounted for an up to 78-fold increase compared to controls (Table 1). A similar trend was observed with regard to the lipopeptide fusaristatin A (9),15 which increased from 10.05 mg in axenic F. tricinctum to 79.40 mg per flask in cocultures. For lateropyrone (5),16,17 however, which is another typical constituent of F. tricinctum, no clear induction was detected (Table 1), indicating that the effects of coculturing are not uniform for all fungal compounds. In addition to the strong increase of constitutively present compounds (with the notable exception of lateropyrone), four further compounds (1−4) that were only detected in cocultures of F. tricinctum and B. subtilis were isolated during this study. These compounds included three new natural products (1, 3, and 4). Compound 1 was isolated as a colorless powder, whose molecular formula was determined as C13H12O4 by HRESIMS (m/z 233.0874 [M + H]+), requiring 8 degrees of unsaturation. The 1H NMR spectrum showed the presence of three aromatic protons at δH 6.30 (2H, d, J = 2.1 Hz, H-9/H-13) and 6.28 (1H, t, J = 2.1 Hz, H-11), two olefinic protons at δH 5.99 (1H, d, H-5) and 5.98 (1H, m, H-3), one methylene group at δH 3.69 (2H, s, H2-7), and one methyl group at δH 2.22 (3H, d, J = 0.5 Hz, H3-14). The 13C NMR spectrum displayed 13 carbon signals (Table 2) including seven protonated carbon signals at 1

from H-5 to C-3 were discerned. Moreover, the HMBC correlations (Table 2) from H3-14 to C-2 (δC 166.5), from H-3 to C-2 and the carbonyl (C-4, δC 179.7), from H-5 to C-4, C-6 (δC 168.5), and C-7, and from H2-7 to C-6 and C-5 allowed the assignment of a 2-methyl and 6-methylene substituted 4pyranone ring. Therefore, compound 1 is identified as 2-(3,5dihydroxybenzyl)-6-methyl-4H-pyran-4-one, which is structurally related to the fungal metabolites macrocarpons A and B, which are produced by the fungi of the family Xylariaceae.18,19 The trivial name macrocarpon C is given to this new compound. Compound 3 exhibited a pseudomolecular peak at m/z 195.06067 [M + H]+ in the HRESIMS spectrum, corresponding to the molecular formula C9H9NO4. The 1H NMR spectrum of 3 revealed four aromatic proton signals at δH 7.91 (1H, dd, H-6), 7.36 (1H, ddd, H-4), 6.62 (1H, ddd, H-5), and 6.61 (1H, dd, H-3), which were assignable to an ABCD spin system and showed a singlet integrated as 2H at δH 4.01 (H2-8) for a methylene group. The 13C NMR and DEPT spectra (Table 3) revealed the presence of one aromatic ring Table 3. 1H and 13C NMR Data and HMBC Correlations of 3 (CD3OD) position 1 2 3 4 5 6 7 8 9

13

Table 2. H and C NMR Data and HMBC Correlations of 1 (acetone-d6) position 2 3 4 5 6 7 8 9 10 11 12 13 14 a

δC, type 166.5, 114.3, 179.7, 114.3, 168.5, 40.1, 138.9, 108.3, 159.7, 102.2, 159.7, 108.3, 19.6,

C CH C CH C CH2 C CH C CH C CH CH3

δH mult. (J in Hz)

HMBC correlations (H→C)

5.98 m

C-2, C-14, C-5, C-4wa

5.99 d (2.2)

C-4w, C-6, C-7, C-3

3.69 s

C-5, C-6, C-8, C-9/C-13

6.30 d (2.1)

C-7, C-10, C-11, C-13

6.28 t (2.1)

C-9/C-13, C-10/C-12

6.30 d (2.1) 2.22 d (0.5)

C-7, C-9, C-11, C-12 C-2, C-3

δC, type 112.3, 151.9, 112.5, 135.8, 116.4, 133.4, 171.9, 45.5, 174.1,

C C CH CH CH CH C CH2 C

δH mult. (J in Hz)

HMBC correlations (H→ C)

6.61 dd (8.7, 1.0) 7.36 ddd (8.7, 7.2, 1,6) 6.62 ddd (7.8, 7.2, 1,0) 7.91 dd (7.8, 1.6)

C-5, C-6, C-3, C-7,

C-1 C-2 C-1 C-2, C-4

4.01 s

C-2, C-9

(δC 151.9, 135.8, 133.4, 116.4, 112.5, 112.3), one methylene (δC 45.5, C-8), and two carbonyl groups (δC 174.1, 171.9). One carbonyl (δC 171.9, C-7) was located at C-1 of the benzene ring, since H-6 (δH 7.91, dd) showed an HMBC correlation with the former. The methylene group (H2-8) showed HMBC correlations only to C-2 (δC 151.9) and the second carbonyl (δC 174.1, C-9), while no correlation from H-3 to the methylene group (C-8) was observed. Thus, C-2 and C-8 were connected through a nitrogen atom, and two carboxylic groups were present at C-7 and C-9 to complete the structure of 3. Compound 3 was thus identified as N-(carboxymethyl)anthranilic acid. This compound was first introduced as a synthetic compound in 1890 by Heumann as a starting material for indigo synthesis.20 Since then, it has been used in many organic syntheses as a starting material.20−22 However, to the best of our knowledge, this is the first report of the isolation of 3 from nature. Biosynthetically, compound 3 is an anthranilic acid derivative, which could originate from condensation of anthranilic acid and acetyl Co-A. Anthranilic acid is a key building block of the peptides talaromins A and B produced by the fungus Talaromyces wortmannii23 and of several alkaloids derived from fungi, such as fumiquinazoline A,24 chaetominine,25 asperlicin,26 and cottoquinazoline A.27 On the other hand, anthranilic acid was also reported as a natural product produced by Streptomyces sp.,28,29 Paenibacillus polymyxa,30 and other bacteria.31 Hence, the producer of compound 3 during cocultivation of F. tricinctum and B. subtilis is still unclear.

w: weak correlation.

δC 108.3 (CH-9/CH-13), 102.2 (CH-11), 114.3 (CH-3/CH5), 40.1 (CH2-7), and 19.6 (CH3-14) and six quaternary carbon signals that were distinguished by DEPT-135 and HSQC experiments. The chemically equivalent aromatic protons (H9/H-13) showed meta coupling to the third aromatic proton (H-11) and long-range correlation to H2-7 in the COSY spectrum, thus indicating the presence of a symmetrically trisubstituted benzene ring. The HMBC correlations from H2-7 to CH-9/CH-13 and C-8 (δC 138.9) and from H-9/H-13 to C10/C-12 (δC 159.7), CH-11, and CH2-7 further suggested a 10,12-dihydroxy-8-methylenebenzene ring being present in 1. In the COSY spectrum, additional long-range correlations via four bonds were observed between H-3/H-5 and H-3/H3-14. This was corroborated by the HMBC spectrum, in which the correlations from H3-14 to C-3, from H-3 to C-14 and C-5, and C

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Antibiotic abbreviations and breakpoints for resistance were applied according to the CLSI guidelines:43 AMX, amoxicillin; CAZ, ceftazidime; CHL, chloramphenicol; CIP, ciprofloxacin; CLA, clarithromycin; CLI, clindamycin; CPM, cefepime; ERY, erythromycin; FOS, fosfomycin; GEN, gentamycin; KAN, kanamycin; MER, meropenem; MXF, moxifloxacin; NIT, nitrofurantoin; PIP/TAZ, piperacillin/tazobactam; TEL, telithromycin; TET, tetracycline. bCompound 5 is hardly soluble in aqueous solvents, methanol, ethanol, and the used dimethyl sulfoxide; variations in outcome may be caused by this low solubility, and antimicrobial activity of 5 may be even higher if compound solubility can be improved. cThe experiment was repeated six times and showed variations in the depicted ranges. dR: resistant. eMRSA: methicillin-resistant S. aureus. fVRE: vancomycin-resistant enterococci.

a

64 0.5 32 16 0.006 16 64 >64 >64 >64 >64 >64 4 >64 >64 8 >64 >64 >64 >64 >64 8 >64 >64 >64 >64 >64 >64 >64 >64 >64 >64 >64 CLAR, ERYR, MXFR, TELR (VREf) susceptible CAZR, CIPR, CPMR, GENR, IMIR, MERR, PIP/TAZR

>64 >64 >64

0.0625 0.25 0.125 >64 2−4c 4 16 4−8c >64 >64

S. pneumoniae ATCC 49619 E. faecalis UW 2689 E. coli ATCC 25922 P. aeruginosa B 63230

>64

>64

0.06 0.125−0.0625 8 0.125 0.5 0.5 >64 >64 >64 8 4−8c 4−8c 16 8 8 >64 >64 >64 8 2−4c 2−4c >64 >64 >64 >64 >64 >64 >64 >64 >64 >64 >64 >64

susceptible susceptible AMXR,d CHLR, CIPR, CLIR, ERYR, FOSR, GENR, KANR, NITR, TETR (MRSAe) susceptible B. subtilis 168 trpC2 S. aureus ATCC 29213 S. aureus 25697

2 1 resistance phenotypea tested organism

Table 4. Minimal Inhibitory Concentration (MIC) [μg/mL] of the Isolated Compounds

3

4

5b

6

7

8

9

vancomycin

ciprofloxacin

tetracycline

Compounds 2 and 4 were likewise lacking either in axenic F. tricinctum or in B. subtilis. Compound 2 was identified as (−)-citreoisocoumarin by comparison of the spectroscopic data with the literature.32,33 The NMR, MS, and UV spectra of compound 4 matched well with those reported for citreoisocoumarinol;33 however, their optical rotation values were opposite ([α]D −20.1 (4); +20.2 (lit.)33); thus 4 was determined as (−)-citreoisocoumarinol, which is a new natural product. Recently, the production of 2 was reported from F. graminearum, F. tricintum, and other Fusarium species as presumably from a redirected pathway of bikaverin due to nitrogen starvation of the fungal cultures.34 Hence, the production of 2 induced by coculture of F. tricinctum and B. subtilis might be a result of the competition for nutrients between fungus and bacterium. The induced accumulation of fungal metabolites as reported in this study was found to correlate with the time of preincubation of the solid rice medium with B. subtilis prior to inoculation with F. tricinctum. When the fungus and the bacterium were inoculated on rice medium at the same day, no, or only a minor, effect on the accumulation of the fungal products was observed (data not shown). The strongest induction of fungal metabolites was detected following a sixor eight-day preincubation of the rice medium with B. subtilis, whereas this effect declined again after 10 days of preincubation (data not shown). For the experiments reported in this paper, a six-day preincubation of B. subtilis prior to inoculation with F. tricinctum was chosen. Under these experimental conditions the fungal growth as observed by visual inspection of the culture flasks was initially slowed down compared to axenic controls of F. tricinctum, but recovered after six to eight days of coculture, indicating an inhibitory effect caused by the presence of B. subtilis in the medium. In a second set of experiments F. tricinctum was cocultured with Streptomyces lividans, a bacterial species that occurs in soil35−37 but has also been reported as an endophyte of plants.38 Also here, a clear induction of the accumulation of enniatins B (6), B1 (7), and A1 (8) as well as of fusaristatin A (9) was detected when compared to axenic controls of F. tricinctum (Figure 1). Compounds 1−4, however, were lacking. Instead, other compounds that remain to be identified were detected in extracts of F. tricinctum cocultured with S. lividans that were absent in fungal and bacterial controls (Figure 1). This indicates that the response of F. tricinctum to B. subtilis may at least partly be specific. All isolated compounds were tested for their antibacterial activities (Table 4) against a broad spectrum of important Gram-positive and Gram-negative nosocomial pathogens. Enniatins B1 (7) and A1 (8) were active against B. subtilis 168 trpC2, the bacterial strain used for cocultivation, with MICs of 16 and 8 μg/mL, respectively, while enniatin B (6) did not inhibit the growth of this strain up to the highest concentration tested (64 μg/mL). Interestingly, the biosyntheses of these active enniatins, as opposed to the inactive enniatin B, were particularly upregulated by Fusarium during coculturing, leading to the tempting speculation that the fungus might have initiated their production in order to suppress its competitor. Enniatin B1 (7) further displayed MIC values of 8, 4, and 8 μg/mL against S. aureus, S. pneumoniae, and E. faecalis, respectively, and enniatin A1 (8) showed slightly lower MICs of 4−8, 2−4, and 4 μg/mL, respectively. The constitutively present lateropyrone (5) also displayed potent inhibitory activity against B. subtilis as well as against S. aureus, S.

4 0.25 32

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measuring the OD600 in a Tecan microtiter plate reader (Infinite M200, Tecan). Microbial Material. The endophytic fungus was isolated from fresh, healthy rhizomes of Aristolochia paucinervis collected in January 2006 from the mountains of Beni-Mellal, Morocco, as previously described.10 The bacterial strain panel included antibiotic-susceptible strains: a standard laboratory strain, Bacillus subtilis 168 trpC2;41 the quality control strains of the Clinical Laboratory Standard Institute (CLSI), Staphylococcus aureus ATCC 29213, Streptococcus pneumoniae ATCC 49619, and Escherichia coli ATCC 25922;43 and the following (multi)drug-resistant clinical isolates: Staphylococcus aureus 25697 (AiCuris, Wuppertal, Germany), Enterococcus faecalis UW 2689 (Wolfgang Witte, Robert Koch Institute, Wernigerode, Germany), and Pseudomonas aeruginosa B 63230.42 Cocultivation Experiment of F. tricinctum with B. subtilis 168 trpC2. Growth of fungus and bacteria in coculture for isolation and identification of metabolites was carried out in Erlenmeyer flasks (1 L). The fungal and bacterial strains were cultivated on solid rice media. Twenty-four Erlenmeyer flasks (eight flasks for F. tricinctum alone, eight for coculture of F. tricinctum and B. subtilis, and eight for B. subtilis alone) containing 60 mL of distilled water and 50 g of commercially available milk rice (Milch-Reis, ORYZA) each were autoclaved before inoculating the fungus and the bacterium. B. subtilis was grown in lysogeny broth (LB). An overnight culture of B. subtilis was used to inoculate prewarmed LB medium (1:20), which was then incubated at 37 °C with shaking at 200 rpm to mid exponential growth phase (optical density at 600 nm (OD600) of 0.2− 0.4). A 10 mL amount of the bacterial culture was added to the rice medium, which was further incubated for 6 days at 37 °C. After this preincubation, F. tricinctum grown on malt agar (5 pieces, 1 cm × 1 cm) was added to the rice medium containing bacteria (after 6 days incubation) under sterile conditions. Fungal and bacterial controls were grown axenically on rice medium. Coculture and axenic cultures of F. tricinctum or B. subtilis were kept under static conditions at 23 °C until they reached their stationary phase of growth (2 weeks for controls of F. tricinctum and 3 weeks for cocultures). Then 300 mL of EtOAc was added to the cultures to stop the growth of cells followed by shaking at 140 rpm for 8 h. The cultures were then left overnight and filtered on the following day using a Büchner funnel. The EtOAc was removed under vacuum. Each extract was then dissolved in 50 mL of MeOH, and 20 μL of this was then injected into the analytical HPLC column. Extraction and Isolation. The crude extract of cocultures of F. tricinctum and B. subtilis (2 g) was subjected to vacuum liquid chromatography (VLC) using mixtures of n-hexane−EtOAc followed by CH2Cl2−MeOH as the eluting solvent. Fraction 7, eluted with CH2Cl2−MeOH (9:1), was further purified using Sephadex LH-20 followed by semipreparative HPLC with MeOH−H2O to yield 1 (2 mg), 2 (1.5 mg), 4 (0.8 mg), and 9 (4 mg). Fraction 10, eluted with CH2Cl2−MeOH (3:7), and fraction 12 (eluted with MeOH containing 0.1% TFA) were further purified by semipreparative HPLC using MeOH−H2O as the mobile phase to afford 3 (2.3 mg) and 5 (1.8 mg), respectively. Metabolites 6−8 were identified by comparison with authentic standards employing HPLC and LC-MS. Macrocarpon C (1): white, amorphous powder; UV (λmax, MeOH) log ε 203 (3.5), 251 (3.1) nm; 1H NMR (600 MHz, acetone-d6) and 13 C NMR (150 MHz, acetone-d6) see Table 2; ESIMS m/z 233.2 [M + H]+, 231.3 [M − H]−; HRESIMS m/z 233.08074 [M + H]+ (calcd for C13H13O4 233.08084). N-(Carboxymethyl)anthranilic acid (3): yellow, amorphous powder; UV (λmax, MeOH) log ε 218 (4.1), 255 (3.7), 348 (3.4) nm; 1H NMR (600 MHz, CD3OD) and 13C NMR (150 MHz, CD3OD) see Table 3; ESIMS m/z 196.0 [M + H]+, 194.3 [M − H]−; HRESIMS m/z 196.06067 [M + H] + (calcd for C 9 H 10 NO 4 196.06043). (−)-Citreoisocoumarinol (4): yellow, amorphous powder; [α]22D −20.1 (c 0.09, MeOH); ESIMS m/z 281.0 [M + H]+, 279.4 [M − H]−; UV and NMR data matched well with the published data.33 Antibacterial Assay. MIC values were determined by the broth microdilution method according to CLSI guidelines.43 For preparation

pneumoniae, and E. faecalis with MICs of 8, 2−4, 4−8, and 8 μg/mL, respectively. All other metabolites isolated in this study were inactive against all of the strains, tested up to a concentration of 64 μg/mL. A further notable aspect is that all antibacterial compounds described in this study retained full activity against multidrug-resistant staphylococcal and enterococcal clinical isolates (for details on the resistance phenotypes, see the footnote of Table 4). Lateropyrone (5) had been previously reported to inhibit the growth of S. aureus, although no quantitative value had been determined.16 Regarding fusaristatin A (9), a previous publication had found this compound inactive against S. aureus and Pseudomonas aeruginosa at a concentration of 100 μg/mL,15 which is in accordance with our results. For enniatin B (6), antibacterial activity was previously reported against Mycobacterium tuberculosis,39 Clostridium perf ringens CECT 4647, Salmonella enterica CECT 554, and S. aureus CECT 976.40 In conclusion, coculturing of F. tricinctum and B. subtilis caused a strong enhancement of constitutively present fungal products and also stimulated the accumulation of several compounds that were not detected in either fungal or bacterial axenic controls. Most of these latter compounds (if not all) originate from F. tricinctum based on structural analogies with other known fungal products. Several fungal products identified in this study such as 5, 7, and 8 show antibacterial activity against several Gram-positive bacteria including MRSA. The induced production of compounds 1−4 was not duplicated when F. tricinctum was cocultivated with S. lividans. The latter treatment led rather to an accumulation of other, as yet unknown, compounds that were lacking in cocultures of F. tricinctum and B. subtilis. The fact that cocultivation in the present study enhanced the production of bioactive fungal products that inhibited the growth of the bacterial competitor demonstrates the general value of such cocultivating experiments and encourages further studies, even those including cocultivations with pathogenic bacteria.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were determined on a Perkin-Elmer-241 MC polarimeter. 1D and 2D NMR spectra were recorded on an Avance DMX 600 NMR spectrometer. Chemical shifts were referenced to the residual solvent peak at δH 3.31 (CD3OD) and 2.05 (acetone-d6) for 1H and δC 49.15 (CD3OD) and 29.92 (acetone-d6) for 13C, respectively. Mass spectra were measured with a LCMS HP1100 Agilent Finnigan LCQ Deca XP Thermoquest, and high-resolution electrospray ionization mass spectroscopy (HRESIMS) were recorded with a UHR-TOF maXis 4G (Bruker Daltonics, Bremen) mass spectrometer. HPLC analysis was performed with a Dionex P580 system coupled to a photodiode array detector (UVD340S); routine detection was at 235, 254, 280, and 340 nm. The separation column (125 × 4 mm) was prefilled with Eurosphere-10 C18 (Knauer, Germany), and the following gradient was used (MeOH−H2O (containing 0.1% HCOOH)): 0−5 min (10% MeOH); 5−35 min (10−100% MeOH); 35−45 min (100% MeOH). Semipreparative HPLC was performed using a Merck Hitachi HPLC System (UV detector L-7400; pump L-7100; Eurosphere-100 C18, 300 × 8 mm, Knauer, Germany). Column chromatography was performed on Silica gel 60 M (230−400 mesh ASTM, Macherey-Nagel GmbH & Co. KG, Dueren, Germany) and Sephadex LH-20 (Sigma). TLC was carried out on precoated silica gel plates (silica gel 60 F-254, Merck KGaA, Darmstadt, Germany) for monitoring of fractions by using EtOAc−MeOH−H2O (30:5:4) and CH2Cl2−MeOH (9:1) as solvent systems. Detection was at 254 and 366 nm or by spraying the plates with anisaldehyde reagent. Bacterial growth was monitored by E

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of the inoculum, the direct colony suspension method was used with an inoculum of 5 × 105 colony forming units/mL after the last dilution step. Compounds were added from stock solution (10 mg/mL in DMSO), resulting in a final DMSO amount of 0.64% at the highest antibiotic concentration tested (64 μg/mL). Serial 2-fold dilutions of antibiotics were prepared with DMSO being diluted along with the compounds.

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ASSOCIATED CONTENT

* Supporting Information S

1

H, 13C, and MS spectra for compounds 1 and 3 are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*Phone: +49 211 81 14187. Fax: +49 211 81 11923. E-mail: [email protected]. *Phone: +49 211 81 14163. E-mail: proksch@uni-duesseldorf. de. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by grants of the BMBF to P.P., by the German Research Foundation (FOR854, BR 3783/1-2) to H.B.-O., and by the iGRASPseed grant from the Research Center Juelich (Germany) and the University of Duesseldorf (Germany) to D.T. A.R.B.O. thanks DAAD for a doctoral scholarship. Technical assistance of H. Goldbach-Gecke is gratefully acknowledged. In addition, we thank H.-G. Sahl (University of Bonn, Germany), W. Witte (Robert-Koch Institute, Wernigerode, Germany), and AiCuris GmbH & Co. KG (Wuppertal, Germany) for providing clinical isolates.

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REFERENCES

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dx.doi.org/10.1021/np400589h | J. Nat. Prod. XXXX, XXX, XXX−XXX